The role of DNA methylation in chondrogenesis of human iPSCs as a stable marker of cartilage quality.

BACKGROUND: Lack of insight into factors that determine purity and quality of human iPSC (hiPSC)-derived neo-cartilage precludes applications of this powerful technology toward regenerative solutions in the clinical setting. Here, we set out to generate methylome-wide landscapes of hiPSC-derived neo-cartilages from different tissues-of-origin and integrated transcriptome-wide data to identify dissimilarities in set points of methylation with associated transcription and the respective pathways in which these genes act. METHODS: We applied in vitro chondrogenesis using hiPSCs generated from two different tissue sources: skin fibroblasts and articular cartilage. Upon differentiation toward chondrocytes, these are referred to as hFiCs and hCiC, respectively. Genome-wide DNA methylation and RNA sequencing datasets were generated of the hiPSC-derived neo-cartilages, and the epigenetically regulated transcriptome was compared to that of neo-cartilage deposited by human primary articular cartilage (hPAC). RESULTS: Methylome-wide landscapes of neo-cartilages of hiPSCs reprogrammed from two different somatic tissues were 85% similar to that of hPACs. By integration of transcriptome-wide data, differences in transcriptionally active CpGs between hCiC relative to hPAC were prioritized. Among the CpG-gene pairs lower expressed in hCiCs relative to hPACs, we identified genes such as MGP, GDF5, and CHAD enriched in closely related pathways and involved in cartilage development that likely mark phenotypic differences in chondrocyte states. Vice versa, among the CpG-gene pairs higher expressed, we identified genes such as KIF1A or NKX2-2 enriched in neurogenic pathways and likely reflecting off target differentiation. CONCLUSIONS: We did not find significant variation between the neo-cartilages derived from hiPSCs of different tissue sources, suggesting that application of a robust differentiation protocol such as we applied here is more important as compared to the epigenetic memory of the cells of origin. Results of our study could be further exploited to improve quality, purity, and maturity of hiPSC-derived neo-cartilage matrix, ultimately to realize introduction of sustainable, hiPSC-derived neo-cartilage implantation into clinical practice.

Evaluation of Right Ventricular Myocardial Properties Using Systolic Myocardial T1 Mapping.

Introduction Myocardial properties can be quantitatively evaluated using myocardial native T1 values (nT1) obtained using cardiac magnetic resonance imaging (CMR). In terms of myocardial wall thickness, the left ventricular nT1 is easy to measure, but the right ventricular nT1 is difficult. Patients with congenital heart disease often develop right ventricular overload. If right ventricular nT1 can be measured consistently, inflammation and fibrosis of the right ventricular myocardium can be quantitatively evaluated. We aimed to determine whether T1 mapping during systole can be used to evaluate right ventricular myocardial properties. Methods T1 mapping was performed at diastole and systole. Systolic T1 mapping was calculated from diastolic T1 mapping and cine images. The myocardial properties of both ventricles were evaluated in 13 healthy volunteers (21-26 years old) and 12 patients with right ventricular overload (12-41 years old) who underwent CMR examination at our hospital. Results From the analysis of left ventricular nT1, we found that nT1 did not change significantly during the cardiac cycle. However, right ventricular nT1 changed between diastole and systole because the right ventricle is affected by blood. Although there was no difference in right ventricular diastolic nT1 between the patients and volunteers (1,346.8 vs. 1,347.6 ms, p = 0.852), the right ventricular systolic nT1 was significantly higher in patients than in volunteers (1,312.7 vs. 1,233.8 ms, p = 0.002). This indicates that right ventricular myocardial damage occurs in patients with right ventricular overload. Conclusion Systolic right ventricular myocardial T1 mapping allows assessment of right ventricular myocardial properties. The right ventricular myocardial systolic nT1 is useful for evaluating myocardial damage due to right ventricular stress and myocardial injury. Measuring right ventricular nT1 may allow consideration of early therapeutic intervention.

Culture system for longitudinal monitoring of bone dynamics ex vivo.

To quantify and visualize both bone formation and resorption within osteochondral explants cultured ex vivo is challenging with the current analysis techniques. An approach that enables monitoring of bone remodeling dynamics is longitudinal microcomputed tomography (µCT), a non-destructive technique that relies on repeated µCT scanning and subsequent registration of consecutive scans. In this study, a two-compartment culture system suitable for osteochondral explants that allowed for µCT scanning during ex vivo culture was established. Explants were scanned repeatedly in a fixed orientation, which allowed assessment of bone remodeling due to adequate image registration. Using this method, bone formation was found to be restricted to the outer surfaces when cultured statically. To demonstrate that the culture system could capture differences in bone remodeling, explants were cultured statically and under dynamic compression as loading promotes osteogenesis. No quantitative differences between static and dynamic culture were revealed. Still, only in dynamic conditions, bone formation was visualized on trabecular surfaces located within the inner cores, suggesting enhanced bone formation towards the center of the explants upon mechanical loading. Taken together, the ex vivo culture system in combination with longitudinal µCT scanning and subsequent registration of images demonstrated potential for evaluating bone remodeling within explants.

A case report of feline mast cell tumour with intertumoral heterogeneity: Identification of secondary mutations c.998G>C and c.2383G>C in KIT after resistance to toceranib.

A 12-year-old male domestic cat with multiple subcutaneous mast cell tumours (MCTs) presented with a 2-week history of pruritus and raw/bleeding skin from self-trauma at Kagoshima University Veterinary Teaching Hospital. Polymerase chain reaction (PCR) and histopathological analyses revealed intertumoral heterogeneity among tumour locations based on the mutation status of KIT. In addition, the expression pattern of KIT was characterized. After failed treatment with vinblastine (2.0-2.2 mg/m2, intravenous administration, two doses in total) or nimustine (25 mg/m2, intravenous administration, two doses in total), toceranib (2.2-2.6 mg/kg, orally administered, every other day) was administered to treat recurrent MCTs harbouring the KIT exon eight internal tandem duplication mutation, achieving a complete response. However, toceranib resistance developed 2 months after treatment initiation. Subsequent PCR analysis was conducted to identify the mutational status of KIT in each MCT and to detect the presence of secondary mutations associated with the acquisition of toceranib resistance. Secondary KIT mutations (c.998G>C and c.2383G>C), which were not initially detected in tumour cells at diagnosis, were identified after the development of resistance to toceranib. This indicates that the tumour cells in feline MCTs in the same case have diverse characteristics. Our findings encourage further investigation into the development of therapeutic strategies for feline MCTs, particularly focusing on the heterogeneous nature of KIT/KIT and overcoming acquired resistance to toceranib.

Cartilage-derived cells display heterogeneous pericellular matrix synthesis in agarose microgels.

The pericellular matrix (PCM) surrounding chondrocytes is essential for articular cartilage tissue engineering. As the current isolation methods to obtain chondrocytes with their PCM (chondrons) result in a heterogeneous mixture of chondrocytes and chondrons, regenerating the PCM using a tissue engineering approach could prove beneficial. In this study, we aimed to discern the behavior of articular chondrocytes (ACs) in regenerating the PCM in such an approach and whether this would also be true for articular cartilage-derived progenitor cells (ACPCs), as an alternative cell source. Bovine ACs and ACPCs were encapsulated in agarose microgels using droplet-based microfluidics. ACs were stimulated with TGF-ß1 and dexamethasone and ACPCs were sequentially stimulated with BMP-9 followed by TGF-ß1 and dexamethasone. After 0, 3, 5, and 10 days of culture, PCM components, type-VI collagen and perlecan, and ECM component, type-II collagen, were assessed using flow cytometry and fluorescence microscopy. Both ACs and ACPCs synthesized the PCM before the ECM. It was seen for the first time that synthesis of type-VI collagen always preceded perlecan. While the PCM synthesized by ACs resembled native chondrons after only 5 days of culture, ACPCs often made less well-structured PCMs. Both cell types showed variations between individual cells and donors. On one hand, this was more prominent in ACPCs, but also a subset of ACPCs showed superior PCM and ECM regeneration, suggesting that isolating these cells may potentially improve cartilage repair strategies.

Wearable sensor systems measure differences in knee laxity between healthy and affected knees during dynamic exercise activities: A systematic review.

Purpose: Knee laxity can be experienced as knee instability which may lead to a limitation in the activity of patients. Current methods to determine knee instability are performed in a static setting, which does not always correlate with dynamic knee laxity during activities. Wearables might be able to measure knee laxity in a dynamic setting and could be of added value in the diagnosis and treatment of excessive knee laxity. Therefore, the aim of this systematic review is to provide an overview of the wearables that have been developed and their ability to measure knee laxity during dynamic activities. Methods: The PRISMA guidelines for systematic reviews were followed. A literature search was conducted in EMBASE, PubMed and Cochrane databases. Included studies assessed patients with knee instability using a non-invasive wearable sensor system during dynamic activity, with comparison to a reference system or healthy knees. Data extraction was performed by two authors via a predefined format. The risk of bias was assessed by The Dutch checklist for diagnostic tests. Results: A total of 4734 articles were identified. Thirteen studies were included in the review. The studies showed a great variety of patients, sensor systems, reference tests, outcome measures and performed activities. Nine of the included studies were able to measure differences in patients with knee instability, all including a tri-axial accelerometer. Differences were not measurable in all parameters and activities in these studies. Conclusions: Wearables, including at least a tri-axial accelerometer, seem promising for measuring dynamic knee laxity in the anterior-posterior and mediolateral direction. At this stage, it remains unclear if the measured outcomes completely reflect the knee instability that patients experience in daily life. Level of Evidence: Level III.

Conventional magnetic resonance imaging key features for distinguishing pathologically confirmed corticobasal degeneration from its mimics: a retrospective analysis of the J-VAC study.

PURPOSE: Due to the indistinguishable clinical features of corticobasal syndrome (CBS), the antemortem differentiation between corticobasal degeneration (CBD) and its mimics remains challenging. However, the utility of conventional magnetic resonance imaging (MRI) for the diagnosis of CBD has not been sufficiently evaluated. This study aimed to investigate the diagnostic performance of conventional MRI findings in differentiating pathologically confirmed CBD from its mimics. METHODS: Semiquantitative visual rating scales were employed to assess the degree and distribution of atrophy and asymmetry on conventional T1-weighted and T2-weighted images. Additionally, subcortical white matter hyperintensity (SWMH) on fluid-attenuated inversion recovery images were visually evaluated. RESULTS: In addition to 19 patients with CBD, 16 with CBD mimics (progressive supranuclear palsy (PSP): 9, Alzheimer's disease (AD): 4, dementia with Lewy bodies (DLB): 1, frontotemporal lobar degeneration with TAR DNA-binding protein of 43 kDa(FTLD-TDP): 1, and globular glial tauopathy (GGT): 1) were investigated. Compared with the CBD group, the PSP-CBS subgroup showed severe midbrain atrophy without SWMH. The non-PSP-CBS subgroup, comprising patients with AD, DLB, FTLD-TDP, and GGT, showed severe temporal atrophy with widespread asymmetry, especially in the temporal lobes. In addition to over half of the patients with CBD, two with FTLD-TDP and GGT showed SWMH, respectively. CONCLUSION: This study elucidates the distinct structural changes between the CBD and its mimics based on visual rating scales. The evaluation of atrophic distribution and SWMH may serve as imaging biomarkers of conventional MRI for detecting background pathologies.

A multipurpose, adolescent idiopathic scoliosis-specific, short MRI protocol: A feasibility study in volunteers.

INTRODUCTION: Visualization of scoliosis typically requires ionizing radiation (radiography and CT) to visualize bony anatomy. MRI is often additionally performed to screen for neural axis abnormalities. We propose a 14-minutes radiation-free scoliosis-specific MRI protocol, which combines MRI and MRI-based synthetic CT images to visualize soft and osseous structures in one examination. We assess the ability of the protocol to visualize landmarks needed to detect 3D patho-anatomical changes, screen for neural axis abnormalities, and perform surgical planning and navigation. METHODS: 18 adult volunteers were scanned on 1.5 T MR-scanner using 3D T2-weighted and synthetic CT sequences. A predefined checklist of relevant landmarks was used for the parameter assessment by three readers. Parameters included Cobb angles, rotation, torsion, segmental height, area and centroids of Nucleus Pulposus and Intervertebral Disc. Precision, reliability and agreement between the readers measurements were evaluated. RESULTS: 91 % of Likert-based questions scored ≥ 4, indicating moderate to high confidence. Precision of 3D dot positioning was 1.0 mm. Precision of angle measurement was 0.6° (ICC 0.98). Precision of vertebral and IVD height measurements was 0.4 mm (ICC 0.99). Precision of area measurement for NP was 8 mm2 (ICC 0.55) and for IVD 18 mm2 (ICC 0.62) for IVD. Precision of centroid measurement for NP was 1.3 mm (ICC 0.88-0.92) and for IVD 1.1 mm (ICC 0.88-91). CONCLUSIONS: The proposed MRI protocol with synthetic CT reconstructions, has high precision, reliability and agreement between the readers for multiple scoliosis-specific measurements. It can be used to study scoliosis etiopathogenesis and to assess 3D spinal morphology.

Left atrial vortex flow and its relationship with left atrial functions in patients with congenital heart disease.

BACKGROUND: Four-dimensional flow magnetic resonance imaging (MRI) enables blood flow visualization. The absence of left atrial vortex flow (LAVF) has been implicated in the development of thrombus formation and arrhythmias. However, the clinical relevance of this phenomenon in patients with congenital heart disease (CHD) remains unclear. This study aimed to unravel the relationship of LAVF with left atrial functions in patients with CHD. RESULTS: Twenty-five participants who underwent cardiac MRI examinations were included (8 postoperative patients with CHD aged 17-41 years and 17 volunteers aged 21-31 years). All participants were in sinus rhythm. Four-dimensional flow MRI (velocity encoding 100 cm/s) assessed the presence of LAVF, and its relationship with left atrial function determined by transthoracic echocardiography was explored. LAVF was detected in 16 patients. Upon classification of the participants based on the presence or absence of LAVF, 94% of participants in the LAVF group were volunteers, while 78% of those in the without LAVF group were postoperative patients. Participants without LAVF had a significantly lower left atrial ejection fraction (61% vs. 70%, p = 0.019), reservoir (32% vs. 47%, p = 0.006), and conduit (22% vs. 36%, p = 0.002) function than those with LAVF. CONCLUSIONS: LAVF occurred during the late phase of ventricular systole, and left atrial reservoir function may have contributed to its occurrence. Many postoperative patients with CHD experienced a loss of LAVF. LAVF may indicate early left atrial dysfunction resulting from left atrial remodeling.

Synthesis and immunological evaluation of TLR1/2 ligand-conjugated RBDs as self-adjuvanting vaccine candidates against SARS-CoV-2.

We synthesized and evaluated Pam3CSK4-conjugated receptor binding domain (RBD)/deglycosylated RBD as potential anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidates. Our investigation revealed the critical importance of limiting the number of introduced Pam3CSK4 molecules to the RBD in order to preserve its antigenicity. We also confirmed the harmonious integration of the adjuvant-conjugation strategy with the glycan-shield removal strategy.

Postoperative evaluation of left atrial stiffness in patients with congenital heart diseases.

Left atrial (LA) stiffness is one of the most important factors involved in LA remodeling. LA stiffness has rarely been studied postoperatively in patients with congenital heart diseases (CHDs). This study aimed to evaluate LA stiffness in CHD patients postoperatively. Seventy-five patients (CHDs, 67; controls, 8; age range, 10-41 years) who underwent cardiac catheterization at our institution were included in this study. From the pulmonary artery wedge pressure waveform obtained during cardiac catheterization, the pressure range between the v wave and x trough was determined, and this value was divided by the LA reservoir strain obtained by echocardiography to calculate LA stiffness, as previously reported. LA stiffness was significantly higher in postoperative CHD patients than in controls (0.28 ± 0.17 vs. 0.13 ± 0.03, p = 0.001). Nineteen of 52 (37%) patients in their teens, 8 of 11 (73%) patients in their 20 s, and 1 of 4 (25%) patients aged > 30 years had elevated LA stiffness values. In particular, all patients with an LA reservoir strain < 20% had elevated LA stiffness. In postoperative CHD patients, LA stiffness was frequently elevated from a young age. CHD patients with elevated LA stiffness should be cautious of possible LA dysfunction in the future.

Clinical course of pathologically confirmed corticobasal degeneration and corticobasal syndrome.

The clinical presentation of corticobasal degeneration is diverse, while the background pathology of corticobasal syndrome is also heterogeneous. Therefore, predicting the pathological background of corticobasal syndrome is extremely difficult. Herein, we investigated the clinical findings and course in patients with pathologically, genetically and biochemically verified corticobasal degeneration and corticobasal syndrome with background pathology to determine findings suggestive of background disorder. Thirty-two patients were identified as having corticobasal degeneration. The median intervals from the initial symptoms to the onset of key milestones were as follows: gait disturbance, 0.0 year; behavioural changes, 1.0 year; falls, 2.0 years; cognitive impairment, 2.0 years; speech impairment, 2.5 years; supranuclear gaze palsy, 3.0 years; urinary incontinence, 3.0 years; and dysphagia, 5.0 years. The median survival time was 7.0 years; 50% of corticobasal degeneration was diagnosed as corticobasal degeneration/corticobasal syndrome at the final presentation. Background pathologies of corticobasal syndrome (n = 48) included corticobasal degeneration (33.3%), progressive supranuclear palsy (29.2%) and Alzheimer's disease (12.5%). The common course of corticobasal syndrome was initial gait disturbance and early fall. In addition, corticobasal degeneration-corticobasal syndrome manifested behavioural change (2.5 years) and cognitive impairment (3.0 years), as the patient with progressive supranuclear palsy-corticobasal syndrome developed speech impairment (1.0 years) and supranuclear gaze palsy (6.0 years). The Alzheimer's disease-corticobasal syndrome patients showed cognitive impairment (1.0 years). The frequency of frozen gait at onset was higher in the corticobasal degeneration-corticobasal syndrome group than in the progressive supranuclear palsy-corticobasal syndrome group [P = 0.005, odds ratio (95% confidence interval): 31.67 (1.46-685.34)]. Dysarthria at presentation was higher in progressive supranuclear palsy-corticobasal syndrome than in corticobasal degeneration-corticobasal syndrome [P = 0.047, 6.75 (1.16-39.20)]. Pyramidal sign at presentation and personality change during the entire course were higher in Alzheimer's disease-corticobasal syndrome than in progressive supranuclear palsy-corticobasal syndrome [P = 0.011, 27.44 (1.25-601.61), and P = 0.013, 40.00 (1.98-807.14), respectively]. In corticobasal syndrome, decision tree analysis revealed that 'freezing at onset' or 'no dysarthria at presentation and age at onset under 66 years in the case without freezing at onset' predicted corticobasal degeneration pathology with a sensitivity of 81.3% and specificity of 84.4%. 'Dysarthria at presentation and age at onset over 61 years' suggested progressive supranuclear palsy pathology, and 'pyramidal sign at presentation and personality change during the entire course' implied Alzheimer's disease pathology. In conclusion, frozen gait at onset, dysarthria, personality change and pyramidal signs may be useful clinical signs for predicting background pathologies in corticobasal syndrome.

Automatic generation of subject-specific finite element models of the spine from magnetic resonance images.

The generation of subject-specific finite element models of the spine is generally a time-consuming process based on computed tomography (CT) images, where scanning exposes subjects to harmful radiation. In this study, a method is presented for the automatic generation of spine finite element models using images from a single magnetic resonance (MR) sequence. The thoracic and lumbar spine of eight adult volunteers was imaged using a 3D multi-echo-gradient-echo sagittal MR sequence. A deep-learning method was used to generate synthetic CT images from the MR images. A pre-trained deep-learning network was used for the automatic segmentation of vertebrae from the synthetic CT images. Another deep-learning network was trained for the automatic segmentation of intervertebral discs from the MR images. The automatic segmentations were validated against manual segmentations for two subjects, one with scoliosis, and another with a spine implant. A template mesh of the spine was registered to the segmentations in three steps using a Bayesian coherent point drift algorithm. First, rigid registration was applied on the complete spine. Second, non-rigid registration was used for the individual discs and vertebrae. Third, the complete spine was non-rigidly registered to the individually registered discs and vertebrae. Comparison of the automatic and manual segmentations led to dice-scores of 0.93-0.96 for all vertebrae and discs. The lowest dice-score was in the disc at the height of the implant where artifacts led to under-segmentation. The mean distance between the morphed meshes and the segmentations was below 1 mm. In conclusion, the presented method can be used to automatically generate accurate subject-specific spine models.

Knee instability caused by altered graft mechanical properties after anterior cruciate ligament reconstruction: the early onset of osteoarthritis?

Anterior cruciate ligament (ACL) rupture is a very common knee joint injury. Torn ACLs are currently reconstructed using tendon autografts. However, half of the patients develop osteoarthritis (OA) within 10 to 14 years postoperatively. Proposedly, this is caused by altered knee kine(ma)tics originating from changes in graft mechanical properties during the in vivo remodeling response. Therefore, the main aim was to use subject-specific finite element knee models and investigate the influence of decreasing graft stiffness and/or increasing graft laxity on knee kine(ma)tics and cartilage loading. In this research, 4 subject-specific knee geometries were used, and the material properties of the ACL were altered to either match currently used grafts or mimic in vivo graft remodeling, i.e., decreasing graft stiffness and/or increasing graft laxity. The results confirm that the in vivo graft remodeling process increases the knee range of motion, up to >300 percent, and relocates the cartilage contact pressures, up to 4.3 mm. The effect of remodeling-induced graft mechanical properties on knee stability exceeded that of graft mechanical properties at the time of surgery. This indicates that altered mechanical properties of ACL grafts, caused by in vivo remodeling, can initiate the early onset of osteoarthritis, as observed in many patients clinically.

Biomechanical evaluation of different semi-rigid junctional fixation techniques using finite element analysis.

BACKGROUND: Proximal junctional failure is a common complication attributed to the rigidity of long pedicle screw fixation constructs used for surgical correction of adult spinal deformity. Semi-rigid junctional fixation achieves a gradual transition in range of motion at the ends of spinal instrumentation, which could lead to reduced junctional stresses, and ultimately reduce the incidence of proximal junctional failure. This study investigates the biomechanical effect of different semi-rigid junctional fixation techniques in a T8-L3 finite element spine segment model. METHODS: First, degeneration of the intervertebral disc was successfully implemented by altering the height. Second, transverse process hooks, one- and two-level clamped tapes, and one- and two-level knotted tapes instrumented proximally to three-level pedicle screw fixation were validated against ex vivo range of motion data of a previous study. Finally, the posterior ligament complex forces and nucleus pulposus stresses were quantified. FINDINGS: Simulated range of motions demonstrated the fidelity of the general model and modelling of semi-rigid junctional fixation techniques. All semi-rigid junctional fixation techniques reduced the posterior ligament complex forces at the junctional zone compared to pedicle screw fixation. Transverse process hooks and knotted tapes reduced nucleus pulposus stresses, whereas clamped tapes increased nucleus pulposus stresses at the junctional zone. INTERPRETATION: The relationship between the range of motion transition and the reductions in posterior ligament complex and nucleus pulposus stresses was complex and dependent on the fixation techniques. Clinical trials are required to compare the effectiveness of semi-rigid junctional fixation techniques in terms of reducing proximal junctional failure incidence rates.

Semi-synthetic degradable notochordal cell-derived matrix hydrogel for use in degenerated intervertebral discs: Initial in vitro characterization.

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs.

The Impact of Culture Variables on a 3D Human In Vitro Bone Remodeling Model: A Design of Experiments Approach.

Human in vitro bone remodeling models, using osteoclast-osteoblast cocultures, can facilitate the investigation of human bone remodeling while reducing the need for animal experiments. Although current in vitro osteoclast-osteoblast cocultures have improved the understanding of bone remodeling, it is still unknown which culture conditions support both cell types. Therefore, in vitro bone remodeling models can benefit from a thorough evaluation of the impact of culture variables on bone turnover outcomes, with the aim to reach balanced osteoclast and osteoblast activity, mimicking healthy bone remodeling. Using a resolution III fractional factorial design, the main effects of commonly used culture variables on bone turnover markers in an in vitro human bone remodeling model are identified. This model is able to capture physiological quantitative resorption-formation coupling along all conditions. Culture conditions of two runs show promising results: conditions of one run can be used as a high bone turnover system and conditions of another run as a self-regulating system as the addition of osteoclastic and osteogenic differentiation factors is not required for remodeling. The results generated with this in vitro model allow for better translation between in vitro studies and in vivo studies, toward improved preclinical bone remodeling drug development.

Liver Fibrosis Markers Represent Central Venous Pressure in Post-pubertal Patients With Congenital Heart Disease.

Background Central venous pressure (CVP) is one of the most important hemodynamic parameters in patients with congenital heart disease (CHD). In adults, it is well-known that liver fibrosis markers reflect CVP, but this is not well-understood in children. We investigated the liver fibrosis markers in pediatric CHD patients and their ability to predict CVP. Methods We studied 160 patients who underwent cardiac catheterization in our hospital between January 2017 and December 2020. The levels of the fibrotic markers, including type IV collagen 7s, procollagen type III peptide, and hyaluronic acid, were measured. Results Procollagen type III peptide was markedly elevated in infants younger than one year of age. From one to 15 years of age, it was slightly lower than in the infant group, with a peak at around 10 years of age. In the age group of 16 years and older, most of its values were generally high. Type IV collagen 7s and hyaluronic acid levels were high in infants, with no significant differences at later ages. Procollagen type III peptide and hyaluronic acid showed no significant correlation with CVP in any of the age groups, whereas type IV collagen 7s significantly correlated with CVP in the age group above one year old. Conclusions We found that elevated liver fibrosis markers, particularly type IV collagen 7s, correlated with central venous pressure in CHD patients older than one year. Measurement of liver fibrosis markers may allow the early detection of changes in CVP and liver function in patients with CHD.